Journal of Veterinary Behavior 17 (2017) 6e13

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Journal of Veterinary Behavior

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Ovine Research

Effect of multiple environmental stressors on the adaptive capability

of Malpura rams based on physiological responses in a semi-arid
tropical environment
Veerasamy Sejian a, b, c, *, Davendra Kumar a, John B. Gaughan c, Syed M.K. Naqvi a
a
ICAR-Central Sheep and Wool Research Institute, Avikanagar, Rajasthan, India
b
ICAR-National Institute of Animal Nutrition and Physiology, Bangalore, India
c
School of Agriculture and Food Sciences, The University of Queensland, Gatton, Queensland, Australia

a r t i c l e i n f o a b s t r a c t

Article history: A 45-day study was conducted where the primary objective was to evaluate the effect of simultaneously
Received 5 July 2016 imposed multiple stressors (thermal, nutritional, and walking) on the adaptive capability of Malpura
Received in revised form rams based on changes in ingestive behavior, physiological, blood biochemical, and endocrine responses.
6 September 2016
Twenty adult Malpura rams (average body weight 44.9 kg) were used in the study. The rams were
Accepted 28 October 2016
Available online 10 November 2016
divided into 2 groups, CON (n ¼ 10; control) and multiple stressors (MS; n ¼ 10; multiple stressors). All of
the rams were stall fed with a diet consisting of 70% roughage and 30% concentrate. The CON rams were
maintained in a shed under ambient conditions with ad libitum feeding without walking, whereas MS
Keywords:
adaptation
rams were subjected to multiple stressors (thermal, nutritional, and walking). The feed intake (P < 0.01)
cortisol was lower and water intake (P < 0.01) was higher in MS compared to CON rams. Among the physiological
heat stress measurements, respiration rate afternoon (P < 0.01), pulse rate morning (P < 0.01), rectal temperature
Malpura ram afternoon (P < 0.01), skin temperature afternoon (P < 0.05), and both morning and afternoon scrotum
multiple stress temperature (P < 0.01) increased significantly in MS rams. Both hemoglobin (P < 0.05), packed cell
nutritional stress volume (P < 0.01), increased and plasma glucose (P < 0.01) reduced significantly (P < 0.01) in MS as
respiration compared to CON rams. Among the endocrine parameters, the plasma cortisol increased and plasma
sheep
thyroxin reduced significantly (P < 0.05) in MS rams. The study indicates that Malpura rams have the
walking stress
capability to adjust their physio-biochemical and endocrine responses to cope with multiple stressors in
a hot semi-arid environment. Furthermore, the study also indicated that respiration rate, rectal tem-
perature, scrotal temperature, hemoglobin, packed cell volume, and cortisol may act as ideal biological
markers for quantifying the impact of multiple stressors in Malpura rams.
Ó 2016 Elsevier Inc. All rights reserved.

Introduction facing animal science is the need to increase production in the

Livestock production can be adversely affected by the detri-
mental effects of extreme climatic conditions. Climate change is
considered to be the major threat to the viability and sustainability
of livestock production systems in many regions of the world
(Gaughan et al., 2009). A considerable population of poor people
depends on animals for food, fiber, income, social status, security,
and companionship. Hence, one of the biggest challenges currently
* Address for reprint requests and correspondence: Veerasamy Sejian, Animal
Physiology Division, National Institute of Animal Nutrition and Physiology, Adugodi,
Bangalore 560030, India. Tel: þ91-9740726121; Fax: þ91-080-25711420.
E-mail address: drsejian@gmail.com (V. Sejian).
context of climate change. The challenge is exacerbated because
high production animals are subjected to greater influences by
climatic factors, particularly those raised under tropical conditions
(Martello et al., 2010).
High ambient temperature affects the ability of sheep to dissi-
pate body heat, and as a result, respiration rate, body temperature,
heart beat, and water consumption increase (Marai et al., 2007).
Increased body temperature and respiration rate are the major in-
dicators of heat stress in sheep (Al-Haidary, 2004). Furthermore, an
increase in body temperature is usually associated with marked
reductions in feed intake, redistribution in blood flow, and changes
to endocrine functions that will negatively affect the production
and reproductive performance of sheep (Marai et al., 2007).

http://dx.doi.org/10.1016/j.jveb.2016.10.009
1558-7878/Ó 2016 Elsevier Inc. All rights reserved.
 V. Sejian et al. / Journal of Veterinary Behavior 17 (2017) 6e13
During stressful conditions, various endocrine responses are
invoked in an attempt to improve the biological fitness (reduce the
impact of the stressor) of the individual. The front-line hormones to
overcome stressful situations are glucocorticoids and thyroid hor-
mones. The secretion of glucocorticoids is a classic endocrine
response to stress (Kannan et al., 2000). Currently, it appears that
glucocorticosteroids provide an initial integrating signal which in
conjunction with other hormones and paracrine secretions may
determine specific behavioral, physiological, and biochemical re-
sponses in an animal to allow some degree of adaptation when the
animal is exposed to different environmental conditions (Wingfield
and Kitaysky, 2002). The thyroid gland is one of the most sensitive
organs of the ambient heat variation (Rasooli et al. 2004). The
appropriate thyroid gland function and the activity of thyroid
hormones are considered crucial if the productive performance in
domestic animals is to be maintained (Todini, 2007). When the
animals suffer due to the heat load, food ingestion is reduced and
metabolism slows down, resulting in a hypofunction of the thyroid
gland (McManus et al., 2009). Hence, measuring metabolic hor-
mones such as thyroid hormones will give an indication of the
mechanisms of adaptation.
Sheep in hot semi-arid environment are for the most part reared
in extensive systems. The productive potential of sheep in these
areas is influenced by their exposure to harsh climatic factors
(Sejian et al., 2011). Sheep grazing in this ecological zone faces
extreme fluctuations in the quantity and quality of feed on offer
year round (Martin et al., 2004). However, in the context of climate
change, it is not only the heat stress that affects livestock. Other
important environmental stressors include nutritional stress and
walking stress which comes about due to the need to walk long
distances to source adequate feed and water. Most of the produc-
tivity losses of livestock during the summer are incurred through
low pasture availability (Sejian et al., 2013).
Therefore, from climate change perspectives, it is essential to
study the influence of all the major environmental stresses simul-
taneously (Sejian et al., 2013) to understand in depth the adaptive
capability of the target species, in this case sheep. Gaining this
understanding may pave the way for identification of the ideal
requirements for sheep to counteract such environmental ex-
tremes. Hence, an attempt has been made in this study to deter-
mine the effect of multiple stressors that are simultaneously
imposed on sheep. The primary objective of the study presented
here was to evaluate the simultaneous impact of multiple envi-
ronmental stressors (thermal, nutritional, and walking) on the
adaptive capability based on changes in ingestive behavior, physi-
ology, blood biochemical, and endocrine responses in Malpura
rams.
Materials and methods
This study was approved by the Central Sheep and Wool
Research Institute animal ethics committee.
Site of study
The experiment was carried out at the Central Sheep and Wool
Research Institute, which is located in the semi-arid region of India
at longitude 75 280 E, latitude of 26 260 N at an altitude of 320 m
above mean sea level. The average annual minimum and maximum
ambient temperature ranges from 6 C to 46 C. The mean annual
relative humidity ranges from 20% to 85%. The annual rainfall in this
area ranges from 200 to 400 mm with an erratic distribution
throughout the year.
7
Experimental design
Twenty adult Malpura rams (2-4 years old) with mean body
weight of 44.9  0.69 kg were used in a 45-day study. The Malpura
is a triple purpose, hardy sheep breed, which originated in the arid
and semi-arid areas of Western tropical India. The rams were
divided into 2 groups, CON (n ¼ 10; control) and MS (n ¼ 10;
multiple stresses). The animals were housed in asbestos-roofed,
dirt floor sheds 2.4 m high at the center and 1.73 m high at the
sides. The 3.66  7.32 m shed has a holding capacity for 100 adult
rams. A wire mesh partition was used to hold the experimental
animals at a stocking density of 3 m2/animal. The 4 sides of the shed
were covered with open-type wire mesh. The sheep were individ-
ually restrained while feeding to determine individual feed and
water intake. Before the start of the experiment, these animals were
acclimatized to the restraint. At 9 AM, both groups of rams were
removed from the shed. The MS rams were subjected to heat and
walking stress, whereas CON rams were housed in a similar adja-
cent shed. The CON rams were maintained in the shed under
ambient conditions (maximum ambient temperature exposed was
38 C) for the 45 days of the study. The CON rams had ad libitum
access to feed and water. The MS rams were subjected to multiple
stressors each day: thermal, walking, and nutritional. For 18 hours
of each day, the MS rams were housed in the same shed as the CON
rams. The first stressor was exposed to outdoor ambient conditions
for 6 hours each day (10 AM to 4 PM) where the temperature ranged
from 39 C to 44 C. During this 6-hour period, MS animals did not
have access to shade, feed, or water. The second stressor was
walking. The MS rams were subjected to walking stress by walking
them for 14 km. This 14 km was covered in 2 spans. The animals
took 1 hour and 30 minutes (10 AM to 11:30 AM) to complete 1 span
(7 km) and the second span (7 km) was between 2 PM and 3:30 PM. A
face mask was attached to the rams to prevent them grazing while
walking. Before start of the experiment, the animals were accli-
matized to these face masks (Sejian et al., 2012) to avoid any undue
restraining stress. The third stressor was nutritional. Apart from the
prevention of grazing while walking, the MS rams were only fed at
30% of the ad libitum intake of the CON rams.
Feed sample analysis
All the rams were stall fed a diet consisting of 70% roughage and
30% concentrate. The composition of the diet was roughage (Cenchrus
ciliaris) and concentrate mixture (barley 65%, groundnut cake 32%,
mineral mixture 3%, and common salt 1%). Table 1 describes the di-
etary composition of the feed provided to the sheep. The crude
protein of the feed sample was determined by Kjeldahl technique
(AOAC, 1995). Neutral detergent fiber was determined by the Van
Soest et al. (1991) without sodium sulfite or amylase, whereas acid
Table 1
The chemical composition, energy, and nutrient contents of the diet provided to the
animals
Nutrient contents Roughage Concentrate
Ingredients Cenchrus Barley, 650 g/kg; groundnut cake,
ciliaris 320 g/kg; minerals 30 g/kg including
10 g/kg NaCl
Dry matter (%) 92.7 93.3
Crude protein (%) 8.4 14.0
Ether extract (%) 1.9 3.2
Neutral detergent fiber (%) 72.6 48.5
Acid detergent fiber (%) 54.7 9.0
Acid detergent lignin (%) 20 6.23
Digestible energy (MJ) 5.7 12.1
Metabolizable energy (MJ) 4.7 10.1
8 V. Sejian et al. / Journal of Veterinary Behavior 17 (2017) 6e13
detergent fiber and acid detergent lignin were determined according
to method as described by Robertson and Van Soest (1981). The en-
ergy values of the diet are estimated as per the following formulae:
Total digestible nutrient ¼ digestible crude protein/kg þ
digestible carbohydrate/kg þ 2.25  digestible ether extract ¼ 0.4
kg total digestible nutrient where digestible carbohydrate ¼ (crude
fiber þ nitrogen free extract).
Digestible energy (DE) and metabolizable energy (ME) are
calculated as per the following formulae:
DEðMJ=kgÞ ¼ 18:4096  0:4 ¼ 7:36
MEðMJ=kgÞ ¼ 7:36  0:82 ¼ 6:04
Climatic data
Table 2 describes the cardinal weather parameters during the
study period. The minimum and maximum temperatures, dry and
wet bulb temperatures, and relative humidity were measured using
a digital thermohygrometer (Zeal, London, UK). The weather pa-
rameters were recorded twice daily at 7 AM and 2 PM.
Blood collection and plasma separation
Blood (5 mL) was collected at 2 PM on day 0, 15, 30, and 45 from
each animal in the CON and MS treatment groups. Blood was
collected from the jugular vein using 20 gauge-sterilized needles
and plastic syringe in tubes with heparin anticoagulant. Plasma was
separated by centrifugation at 1870 g at room temperature for
20 minutes. The plasma was then divided into equal aliquots in
microcentrifuge tubes and kept frozen at 20 C until further
analysis. Plasma samples were used to determine plasma glucose,
cortisol, triiodothyronine (T3), and thyroxin (T4) concentration.
Variables studied
Feed intake (FI) and water intake (WI) were recorded daily. The
following parameters were measured at 15-day intervals: respira-
tion rate (RR), pulse rate (PR), rectal temperature (RT), skin tem-
perature, scrotum temperature, skin sweating rate, scrotum
sweating rate, hemoglobin (Hb), and packed cell volume (PCV).
Physiological responses were recorded twice daily at 8 AM and 2 PM.
The RR (breaths/min) was recorded by counting flank move-
ments/minute with the help of a stop watch, from a distance of 4-
5 m without disturbing the rams. The PR (beats/min) was
measured by palpating the femoral artery. For recording the pulse
rate, rams were restrained gently. The RT (oC) was recorded using
a clinical thermometer by inserting the thermometer by 6-7 cm
inside the rectum inclined toward the wall of the rectum. RT was
recorded by gently restraining the rams. The skin (flank region)
and scrotum temperatures (oC) were recorded using a noncontact
infrared thermometer (B.S.K. Technologies, Hyderabad, India) by
Table 2
Climatological data during study period both inside and outside the shed
Environment Time of recording Minimum Maximum
temperature ( C) temperature ( C)
Inside the shed Morning (8 AM) 32.41  0.52 33.61
Afternoon (2 PM) 36.50  0.50 38.25
Outside the shed Morning (8 AM) 34.33  0.30 35.12
Afternoon (2 PM) 39.23  0.68 42.58
RH, relative humidity.
maintaining a distance of 5-15 cm. Aim the region where the
temperature has to be taken, press the button on the device, the
temperature is displayed immediately on the screen of the device.
Sweating rate was recorded at weekly intervals at 2 PM. Sweating
rate was measured by method as described by Berman (1957),
based on the time taken for the chromatography paper disc
impregnated with cobalt chloride to change color from violet to
bright rose.
The Hb and PCV were estimated using whole blood samples by
methods as described by Balasubramaniam and Malathi (1992) and
Jain (1986), respectively. Plasma glucose (Tietz, 1976) was estimated
using Span diagnostic kits, India as per standard method using the
UVevisible recording spectrophotometer (UV-160A; Shimadzu
Corporation, Japan). Hormonal parameters such as cortisol
(analytical sensitivity was 10 nM; the intraassay and interassay
coefficient of variations were 5.8 % and 9.2 %, respectively), thyroxin
(T4) (analytical sensitivity 13 nmol/L; intraassay and interassay
coefficient of variations 5.1 % and 8.6 %, respectively), and triiodo-
thyronine (T3) (analytical sensitivity 0.1 nmol/L; intraassay and
interassay coefficient of variations 3.3% and 8.6%, respectively)
were estimated by radioimmuno assay (RIA) using gamma counter
(PCeRIA MAS; Stretec, Germany) using RIA kits supplied by
Immunotech, Marseille Cedex, France.
Data analysis
The data were analyzed using the general linear model pro-
cedure by multivariate analysis of variance which included the ef-
fects of heat stress, nutritional stress, and combined stressors and
their interactions. Effect of fixed factors, namely treatment (TRT:
control [CON] and multiple stressors [MS]) and days (DAY: longi-
tudinal time over which experiment was carried out on days 0, 15,
30, and 45) and the TRT  DAY interaction of the variables studied
were analyzed. Data were presented as mean  standard error of
the mean, and statistical analysis was carried out using SPSS
software, Version 18.0, SPSS Inc., Chicago. The level of statistical
significance was set at P < 0.05.
Results
Feed and water intake
The effects of multiple stressors on FI and WI are presented in
Table 3. The FI was lower (P < 0.01) in the MS rams compared with
the CON rams on days 15, 30, and 45. Total FI was lower (P < 0.01) in
the MS rams (29.22  0.36 dry matter intake [DMI] g/wk0.75/day)
compared with the CON rams (88.26  0.56 DMI g/wk0.75/day).
However, WI was higher (P < 0.01) in the MS rams (9.37  0.17 L/
DMI kg/day) compared with the CON rams (3.92  0.06 L/DMI kg/
day). Furthermore, DAY influenced both FI and WI (P < 0.01) indi-
cating that the differences between TRT after day 0 persisted over
time. In addition, there were significant TRT  DAY effects (P < 0.01)
on both FI and WI.
Dry bulb Wet bulb RH (%)
temperature ( C) temperature ( C)
 0.33 35.47  0.50 23.15  0.51 35.43  1.47
 0.22 42.57  0.45 24.30  0.42 33.14  1.72
 0.37 32.02  0.38 24.45  0.56 40.60  2.36
 0.67 41.37  0.79 25.86  0.48 30.21  2.65
 V. Sejian et al. / Journal of Veterinary Behavior 17 (2017) 6e13 9

Table 3
Effect of multiple stresses on feed and water intake in Malpura rams

Attributes Days Treatments Day mean Effects

CON MS TRT DAY TRT  DAY

0.75
Feed intake (DMI g/w /day) 15 76.85  0.54 29.26  0.28 53.05  0.41 ** ** **
30 92.32  0.48 28.97  0.43 60.65  0.46
45 95.61  0.65 29.44  0.38 62.52  0.52
Mean 88.26  0.56 29.22  0.36 58.74  0.46
Water intake (L/DMI kg/day) 15 4.72  0.10 9.90  0.09 7.31  0.10 ** ** **
30 3.80  0.05 10.03  0.18 6.92  0.12
45 3.23  0.04 8.18  0.24 5.71  0.14
Mean 3.92  0.06 9.37  0.17 6.65  0.12

CON, control; MS, multiple stresses; TRT, treatment; TRT  DAY, treatment and day interaction.
**P < 0.01.

Physiological responses
The effects of multiple stressors on RR, PR, and RT are pre-
sented in Table 4. Among the physiological responses, treatment
influenced respiration rate afternoon (RRA) (P < 0.01), pulse rate
morning (PRM) (P < 0.01), and rectal temperature afternoon
(RTA) (P < 0.01) between CON and MS groups. The RRA was
higher (P < 0.01) in the MS rams (66.1  2.92 breaths/minute)
compared with the CON rams (58.1  3.92 breaths/minute).
Similarly, RTA was higher (P < 0.01) in the MS rams (39.1 
0.16 C) compared with the CON rams (38.6  0.24 C). However,
PRM was lower (P < 0.01) in the MS rams (52.5  2.15 beats/
minute) compared with the CON rams (58.1  2.07 beats/min).
Among the physiological responses, DAY influenced RRA (P <
0.01), PRM (P < 0.01), PRA (P < 0.01), and RTA (P < 0.05)
indicating that the differences between TRT after day 0 persisted
over time. However, TRT  DAY effects only influenced PRM
(P < 0.01).
The effects of multiple stressors on the skin and scrotum tem-
perature and sweating rate are presented in Table 5. The treatment
influenced skin temperature afternoon (STA) (P < 0.05), scrotum
temperature morning (ScTM) (P < 0.01), and scrotum temperature
afternoon (ScTA) (P < 0.01) between CON and MS groups. The STA
was higher (P < 0.05) in the MS rams (39.0  0.3 C) compared with
the CON rams (38.5  0.3 C). Similarly, ScTA was higher (P < 0.01)
in the MS rams (37.7  0.2 C) compared with the CON rams (37.0 
0.2 C). However, the storm was lower (P < 0.01) in the MS rams
(33.6  0.2 C) compared with the CON rams (34.1  0.2 C).
Furthermore, DAY influenced STM (P < 0.01), STA (P < 0.01), ScTM
(P < 0.01), and ScTA (P < 0.01) indicating that the differences

Table 4
Effect of multiple stresses on respiration rate, pulse rate, and rectal temperature in Malpura rams

Attributes Days Treatments Day mean Effects

CON MS TRT DAY TRT  DAY

RRM (breaths/minute) 0 24.0  1.55 22.8  1.84 23.4  1.70 NS NS NS

15 27.6  3.12 25.6  1.97 26.6  2.55
30 25.7  1.69 20.4  1.51 23.1  1.60
45 22.6  1.89 23.4  1.89 23.0  1.89
Mean 25.0  2.06 23.1  1.80 24.1  1.93
RRA (breaths/minute) 0 56.2  3.99 52.8  4.08 54.5  4.04 ** ** NS
15 57.4  4.13 68.4  2.25 62.9  3.19
30 66.2  4.09 80.6  2.35 73.4  3.22
45 52.6  3.45 62.4  3.01 57.5  3.23
Mean 58.1  3.92 66.1  2.92 62.1  3.42
PRM (beats/minute) 0 62.2  1.65 63.2  1.79 62.7  1.72 ** ** **
15 60.8  2.72 47.8  2.80 54.3  2.76
30 54.4  1.36 46.4  1.48 50.4  1.42
45 54.8  2.53 52.4  2.51 53.6  2.52
Mean 58.1  2.07 52.5  2.15 55.3  2.11
PRA (beats/minute) 0 71.4  1.40 68.6  1.61 70.0  1.51 NS ** NS
15 60.8  2.52 58.8  2.53 59.8  2.53
30 62.4  2.08 62.4  1.36 62.4  1.72
45 60.4  3.29 58.4  2.17 59.4  2.73
Mean 63.8  2.32 62.1  1.92 62.9  2.12
RTM ( C) 0 38.2  0.13 38.0  0.13 38.1  0.13 NS NS NS
15 37.8  0.23 38.0  0.25 37.9  0.24
30 38.2  0.25 38.0  0.28 38.2  0.27
45 38.3  0.34 38.1  0.18 38.2  0.26
Mean 38.1  0.24 38.0  0.21 38.1  0.23
RTA ( C) 0 38.8  0.22 38.9  0.13 38.8  0.18 ** * NS
15 38.5  0.20 39.1  0.19 38.8  0.20
30 38.8  0.24 39.3  0.17 39.1  0.21
45 38.4  0.31 39.1  0.15 38.7  0.23
Mean 38.6  0.24 39.1  0.16 38.9  0.21

CON, control; MS, multiple stresses; NS, nonsignificant; PRA, pulse rate afternoon; PRM, pulse rate morning; RRA, respiration rate afternoon; RRM, respiration rate morning;
RTA, rectal temperature afternoon; RTM, rectal temperature morning; TRT, treatment; TRT  DAY, treatment and day interaction.
*P < 0.05, **P < 0.01.
10 V. Sejian et al. / Journal of Veterinary Behavior 17 (2017) 6e13

Table 5
Effect of multiple stresses on skin and scrotum temperature and sweating rate in Malpura rams

Attributes Days Treatments Day mean Effects

CON MS TRT DAY TRT  DAY


STM ( C) 0 35.0  0.4 34.9  0.2 34.9  0.3 NS ** **
15 35.7  0.2 35.9  0.2 35.8  0.2
30 38.4  0.2 38.9  0.1 38.7  0.2
45 37.5  0.3 35.9  0.2 36.7  0.3
Mean 36.7  0.3 36.4  0.2 36.5  0.3
STA ( C) 0 37.5  0.4 37.2  0.3 37.4  0.4 * ** NS
15 38.1  0.4 38.8  0.2 38.5  0.3
30 39.3  0.3 40.3  0.3 39.8  0.3
45 39.1  0.2 39.6  0.2 39.3  0.2
Mean 38.5  0.3 39.0  0.3 38.7  0.3
ScTM ( C) 0 33.7  0.1 34.0  0.1 33.8  0.1 ** ** **
15 34.0  0.1 34.2  0.1 34.2  0.1
30 35.6  0.2 35.0  0.3 35.3  0.3
45 32.9  0.3 31.3  0.4 32.1  0.4
Mean 34.1  0.2 33.6  0.2 33.8  0.2
ScTA ( C) 0 39.1  0.3 39.2  0.2 39.1  0.3 ** ** NS
15 38.2  0.2 38.7  0.2 38.5  0.2
30 36.8  0.1 37.7  0.3 37.3  0.2
45 34.1  0.2 35.1  0.2 34.6  0.2
Mean 37.0  0.2 37.7  0.2 37.4  0.2
SSR (g/m2/hour) 0 132.9  18.1 129.26  28.8 131.07  23.5 NS NS NS
15 156.0  28.8 192.40  53.1 174.19  40.9
30 141.1  25.6 167.77  49.0 154.42  37.3
45 80.8  34.5 199.65  50.0 140.21  42.3
Mean 127.68  26.7 172.27  45.2 150.0  36.0
ScSR (g/m2/hour) 0 926.0  164.1 1190.0  176.4 1058.0  170.3 NS NS NS
15 1029.0  144.3 1327.0  162.3 1178.0  153.3
30 975.4  44.6 1136.0  60.9 1056.0  52.75
45 1232.0  163.2 1486.0  89.3 1359.0  126.3
Mean 1040.0  129.1 1285.0  122.2 1163.0  125.7

CON, control; MS, multiple stresses; NS, nonsignificant; ScSR, scrotum sweating rate; ScTA, scrotum temperature afternoon; ScTM, scrotum temperature morning; SSR, skin
sweating rate; STA, skin temperature afternoon; STM, skin temperature morning; TRT, treatment; TRT  DAY, treatment and day interaction.
*P < 0.05, **P < 0.01.

between TRT after day 0 persisted over time. However, TRT  DAY
effects only influenced (P < 0.01) STM and ScTM.
Blood biochemical and endocrine responses
The effects of multiple stressors on blood biochemical and
endocrine responses are presented in Table 6. Except for T3, all
blood biochemical and endocrine responses differed significantly
between treatments. Among the blood biochemical responses,
treatment influenced Hb (P < 0.05), PCV (P < 0.01), and plasma
glucose (P < 0.01) between CON and MS groups. The Hb was higher
(P < 0.01) in the MS rams (11.45  0.53 g/dL) compared with the
CON rams (10.42  0.51 g/dL). Similarly, PCV was higher (P < 0.01)
in the MS rams (33.19  0.94%) compared with the CON rams (28.74
 1.96%). However, plasma glucose was lower (P < 0.01) in the MS
rams (42.45  2.13 mg/dL) compared with the CON rams (51.86 
2.32 mg/dL). Among the endocrine responses, treatment influenced
plasma cortisol (P < 0.05) and plasma T4 (P < 0.05) between CON
and MS groups. The plasma cortisol was higher (P < 0.01) in the MS
rams (25.15  2.53 nmol/L) compared with the CON rams (15.66 
2.57 nmol/L). However, plasma T4 was lower (P < 0.01) in the MS
rams (61.05  7.16 nmol/L) compared with the CON rams (78.82 
7.52 nmol/L). Among the blood biochemical and endocrine re-
sponses, DAY influenced only the plasma glucose (P < 0.01) indi-
cating that the differences between TRT after day 0 persisted over
time. However, TRT  DAY effects did not influence any of the blood
biochemical or endocrine responses.
Discussion
The study presented here is of practical relevance as occurrences
of multiple stressors are a common phenomenon in semi-arid
tropical environments. Studying the effect of simultaneously
imposed multiple stressors will provide relevant data that can be
used to establish the impact of climate change on livestock pro-
duction. The results of the current study have shown that multiple
stressors influence feed intake, water intake, physiological re-
sponses, biochemical, and endocrine responses differently when
compared to individual stressors in the rams.
Feed and water intakes are the important parameters for
establishing the adaptive capability of sheep (Minka and Ayo, 2009;
Sejian et al., 2010a). Animals subjected to heat stress attempt to
adapt by reducing their feed intake and increasing their water
intake. The significantly lower feed intake and higher water intake
in the MS group shows the severity of these responses when sheep
are exposed to multiple stressors. It is known that when sheep are
exposed to high ambient temperatures, their ability to dissipate
body heat is reduced, and this results in an increase in respiration
rate, body temperature and consumption of water, and a decline in
feed intake (Marai et al., 2007). Marai et al. (2007) postulated a
reason for reduced feed intake in sheep exposed to heat stress. They
explained that exposure to high environmental temperatures
stimulates the peripheral thermal receptors to transmit suppressive
nerve impulses to the appetite center in the hypothalamus, thereby
causing a decrease in feed intake. The decrease in feed intake re-
sults in less metabolic heat production and this could be viewed as
an adaptive mechanism. Several studies have stated that exposure
of sheep to hot environmental conditions induces a marked in-
crease in water turnover, as well as water intake (Monty et al., 1991;
Ismail et al., 1995; Padua et al., 1997). An increase of water intake
may occur as compensation for a deficit of body water which results
from an increase of evaporation through the respiratory tract and
the skin surface (Minka and Ayo, 2009; Darcan et al., 2008).
Furthermore, the significant influence of experimental days in the
 V. Sejian et al. / Journal of Veterinary Behavior 17 (2017) 6e13 11

present study and the significant interaction between treatments
and experimental days on both feed and water intake indicate the
ability of these animals to adapt to the cumulative stressful
conditions.
The physiological functions of animals, such as RT, RR, and PR,
can favor its survival in a hot climate. Several researchers have
studied physiological adaptation mechanisms such as RT, PR, and
RR in small ruminants (Otoikhian et al., 2009; Phulia et al., 2010;
Sejian et al., 2014). In the present study, the significantly higher
RR and RT in MS group, when the animals were exposed to mul-
tiple stressors during the afternoon when the environmental
temperature was in peak indicate the significance of these 2 pa-
rameters for adaptation in Malpura rams. Both RR and RT have
been shown to be good indicators of thermal stress and may be
used to assess the adversity of the thermal environment (Marai
et al., 2002; Daramola and Adeloye, 2009). Onset and degree of
thermal stress in an animal are best reflected by a rise in rectal
temperature and respiratory dynamics, for example, rapid,
shallow respiration (Al-Haidary, 2004; Marai et al., 2007). The
significantly lower PR during morning in the present study in-
dicates the typical adaptive behavior of desert animals of keeping
themselves cool during the night hours to cope up with the severe
heat stress condition during the day time. Furthermore, the
reduced pulse rate in the MS group may be due to a decrease in the
metabolic rate as a result of restricted feeding of this group. This
view was supported by the findings of several investigators who
have reported that there is a correlation between heart rate and
metabolic heat production (Yamamoto and Ogura, 1985; Barkai
et al., 2002). Aharoni et al. (2003) suggested that heart rate de-
creases during thermal stress as a general effort by the animal to
decrease heat production. This reduction could be achieved by the
animal either by a reduction of feed intake, by a reduction in ac-
tivity, or both (Al-Haidary, 2004).
Skin temperature and scrotal temperature showed significant
variation for treatment, indicating their significance for adaptation
in sheep. Scrotal temperature was much more reliable in assessing
the impact of multiple stressors than the skin temperature. This
difference could be attributed to the wool coat on the body skin as
compared to scrotal skin. Furthermore, the scrotum is an important
thermoregulatory organ in sheep (Marai et al., 2007). Hence, scrotal
temperature has higher significance for assessing the thermo-
tolerant capability of sheep. Both skin and the scrotal sweating
rate did not differ between treatments. This suggests that Malpura
rams relied more on respiratory evaporative cooling mechanisms
than the cutaneous evaporative mechanism. This finding was in
contrast to the previous finding in Malpura ewes where there was a
significant effect of multiple stressors on sweating rate (Sejian et al.,
2013). Therefore, there may be a sex difference in adaptive mech-
anisms in Malpura sheep.
Multiple stressors significantly increased both Hb and PCV. The
reason for this could be severe hemoconcentration as a result of the
imposed multiple stressors. Both Hb and PCV are considered to be
good indicators of stress in farm animals (McManus et al., 2009).
Although the animals had ad libitum access to water, the levels of
Hb and PCV were higher in the multiple stressors group again
highlighting the additive effects of multiple stressors. Generally,
during heat stress, severe dehydration has been reported in live-
stock, which ultimately leads to increased levels of Hb and PCV
(Marai et al., 2007; McManus et al., 2009). Furthermore, severe
water deprivation in the MS rams during walking stress could have
aggravated the condition. The reduced plasma glucose in the MS
treatment could be attributed to both food deprivations as well as

Table 6
Effect of multiple stresses on blood biochemical and endocrine responses in Malpura rams

Attributes Days Treatments Day mean Effects

CON MS TRT DAY TRT  DAY

Hb (g/dL) 0 10.30  0.39 10.33  0.49 10.32  0.44 * NS NS

15 11.10  0.56 12.11  0.86 11.60  0.71
30 10.01  0.71 12.06  0.38 11.03  0.55
45 10.28  0.37 11.32  0.37 10.80  0.37
Mean 10.42  0.51 11.45  0.53 10.94  0.52
PCV (%) 0 32.00  1.02 33.13  1.00 32.16  1.01 ** NS NS
15 26.95  1.98 31.78  1.44 29.36  1.71
30 28.36  3.07 33.65  0.68 31.00  1.88
45 28.46  1.75 34.20  0.63 31.33  1.19
Mean 28.74  1.96 33.19  0.94 30.97  1.45
Glucose (mg/dL) 0 51.42  2.03 48.22  4.26 49.82  3.15 ** ** NS
15 54.12  1.50 44.37  1.65 49.24  1.58
30 49.02  1.86 35.40  1.22 42.21  1.54
45 52.86  3.90 41.82  1.39 47.34  2.65
Mean 51.86  2.32 42.45  2.13 47.15  2.23
Cortisol (nmol/L) 0 14.19  1.77 15.73  3.87 14.96  2.82 * NS NS
15 18.72  2.02 26.43  2.17 22.57  2.10
30 13.71  3.18 28.39  1.63 21.05  2.41
45 16.02  3.29 30.05  2.45 23.03  2.87
Mean 15.66  2.57 25.15  2.53 20.40  2.55
T3 (nmol/L) 0 1.27  0.28 1.30  0.28 1.29  0.28 NS NS NS
15 1.52  0.24 0.97  0.16 1.25  0.20
30 1.62  0.18 0.99  0.14 1.31  0.16
45 1.46  0.21 1.46  0.21 1.46  0.21
Mean 1.47  0.23 1.18  0.20 1.33  0.22
T4 (nmol/L) 0 79.81  9.67 78.41  8.41 79.11  9.04 * NS NS
15 80.34  5.62 54.16  9.00 67.25  7.31
30 76.78  6.91 47.64  6.31 62.21  6.61
45 78.37  7.89 63.99  4.93 71.18  6.41
Mean 78.82  7.52 61.05  7.16 69.94  7.34

CON, control; Hb, hemoglobin; MS, multiple stresses; NS, nonsignificant; PCV, packed cell volume; T3, triiodothyronine; T4, thyroxin; TRT, treatment; TRT  DAY, treatment
and day interaction.
*P < 0.05, **P < 0.01.
12 V. Sejian et al. / Journal of Veterinary Behavior 17 (2017) 6e13
the need to increase utilization of glucose during walking (Sejian
et al., 2012). The decrease in glucose levels could be related to a
decrease in insulin and thyroxine, which are closely associated with
energy metabolism during stress (Rasooli et al., 2004). A decrease in
plasma glucose could also be due to the marked dilution of blood or
increase in the plasma glucose utilization to produce more energy
for greater muscular expenditures required for high muscular ac-
tivity (Rasooli et al., 2004; Sejian et al., 2010b). Nutrient restriction
combined with increased glucose utilization due to increased res-
piratory muscular activity after thermal exposure, resulting in more
reduction in glucose concentration in multiple stressors group.
Multiple stressors significantly increased plasma cortisol con-
centration, indicating that these animals are under stress. However,
the cortisol concentration (25.15 nmol/L) obtained in this study was
much lower than when 2 stressors (51.00 nmol/L) and 3 stressors
(31.03 nmol/L) were simultaneously imposed on Malpura ewes
(Sejian et al., 2010b; Sejian et al., 2013). This shows that Malpura
rams were able to cope with the multiple stressors much better than
Malpura ewes as reflected by minimum increase in plasma cortisol
level to elicit the stress relieving effects as cortisol is thermogenic in
nature which could contribute to additional heat load (Sejian et al.,
2010b). Furthermore, the TRT  DAY also did not influence plasma
cortisol level which was in contrast to the findings of 2 and 3
stressors simultaneously in Malpura ewes indicating that Malpura
rams were able to cope up with the multiple stressors with the
minimum possible increase in cortisol level. Similarly, the effect of
multiple stressors on plasma T3 and T4 were not severe as only T4
differed between the groups. This was again being in contrast to the
previous findings in Malpura ewes (Sejian et al., 2010b; Sejian et al.,
2013) where much higher levels of both T3 and T4 were reported.
This again points toward a sex difference in adaptive mechanisms in
Malpura sheep. The reduced T4 concentration in multiple stressors
group could be attributed to the reduced metabolic activity of these
ewes to suppress heat production. During summer, the exposure of
animals to high ambient temperature was associated with depres-
sion of thyroid activity, thereby causing a relatively lower concen-
tration of thyroid hormones (Rasooli et al., 2004; Stockman et al.,
2011). Besides endogenous and environmental, climatic factors,
nutrition plays a primary role in thyroid gland activity and on blood
thyroid hormone concentrations (Todini, 2007). These effects sug-
gest that energy balance could play a major role in affecting the
decrease in plasma thyroid hormone levels.
Conclusion
The findings from the present study have made a significant
contribution to understanding the intricacies of multiple stressors
on the physiological, blood biochemical, and the endocrine re-
sponses of Malpura rams. The severities of multiple stressors were
determined by the reduced feed and increased water intake in
Malpura rams. Furthermore, the results from the present study
have shown that Malpura rams relied more on respiratory cooling
mechanisms rather than cutaneous evaporative mechanisms to
cope up to multiple stressors. In addition, the study indicates that
Malpura rams have the capability to adjust their physio-
biochemical and endocrine responses to cope with multiple
stressors in the hot semi-arid environment. The study also indi-
cated that RR, RT, scrotum temperature, Hb, PCV, and cortisol may
act as ideal biological markers for quantifying the impact of mul-
tiple stressors in Malpura rams.
Acknowledgments
The authors are highly thankful to Indian Council of Agricultural
Research for funding this project. The authors are also thankful to
the Director of Central Sheep and Wool Research Institute for
providing the research facilities and to Shri K.C. Sharma and Shri
Kamal Kumar for their technical help during the experiment.
The idea of article was conceived and the experiment was
designed by V. Sejian. The experiment was performed by D. Kumar
and V. Sejian. The data were analyzed by Dr. S. M. K. Naqvi. The
article was written by John Gaughan and V. Sejian.
Ethical considerations
This study was approved by the Central Sheep and Wool
Research Institute animal ethics committee.
Conflict of interest
The authors declare that there is no conflict of interest for this
article.
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